This invention relates to a standard whole blood composition, methods for making the composition and methods for determining the potency of blood clotting inhibitory substances.
Fundamental to human life and well being is the ability of human blood, in response to certain stimuli, to thicken and eventually form structures known as blood clots. Blood clotting occurs in response to both external and internal bleeding. Conversely, unwanted clot formation or blood thickening can have undesirable effects among which are circulatory blockages. Certain medical procedures, for example cardiopulmonary bypass (CPB), are obviously adversely affected by blood clotting and would not be possible without means to prevent blood clot formation.
Medical science has developed pharmacological agents which modify the inherent clotting characteristics of human blood. These substances are widely used to treat diseases and to perform medical procedures. A substance of major importance is a naturally occurring material known as heparin. Heparin is a polysaccharide sulfuric acid ester found especially in lung, liver and intestinal tissue and has the ability in certain circumstances to prolong the clotting time of blood. Commercially available heparin is derived from animal tissues principally porcine intestine or bovine lung. As presently understood, commercially processed heparin is a complex substance and its pharmacological activity per unit weight may vary depending on the characteristics of a specific batch of material.
Heparin is sold with its biological potency expressed in U.S.P. units. U.S.P. units are related to the clot formation inhibition of heparin mixed with sheep plasma (blood with all cells removed by centrifugation), The United States Pharmacopeia, 21st Edition (1985) pp. 481-482.
It is well known that there are significant differences in the body's clotting systems (i.e., the blood solids including red blood cells, white blood cells and platelets and the circulatory coagulation factor proteins associated with the plasma) among different individuals. While healthy persons may exhibit blood clotting within so called normal ranges, the actual physiological performance of their system may exhibit wide variations. The production and metabolism of the classical coagulation factors, for example, can vary within fairly wide ranges. One can readily see therefore that care must be used in predicting the response of a given individual to medications affecting coagulation or in developing data based on a blood sample from one donor which can be expected to be applicable to the general population.
Whole blood clotting and plasma clotting involve different mechanisms and substances. Plasma lacks many elements present in whole blood, namely platelets, red blood cells, and white blood cells, which are intricately involved in the hemostatic process. Since heparin is known to interact with these elements, it is readily apparent that the failure of traditional laboratory assays that are typically based on analysis of blood plasma, e.g., the prothrombin time (PT) or the Activated Partial Thromboplastin Time (APTT) test, to monitor heparin anticoagulation effect is due to the inability to duplicate in the lab the true hemostatic status of the patient. Moreover, human blood plasma and sheep blood plasma (used in the U.S.P. assay) are obviously different in their clotting response. Because of these factors, the blood clotting inhibition response of a particular human to a dose of heparin from a particular manufacturer's batch of heparin is somewhat unpredictable.
Medical science has developed various techniques to measure the clotting ability of a sample of human blood. A commonly used procedure is the determination of the blood's Activated Clotting Time (ACT). See, for example, Hattersley, P., JAMA, 196:150-154, 1966; LaDuca F., et al., J. Extra-Corpor. Technol., 19:358-364, 1987; and Dutton et al., Anaesthia, 38:264-68, 1983. In this method the amount of time required for a 0.5 to 2.0 ml sample of the patient's blood to clot is measured. A normal ACT range is 140 to 180 seconds for a population of patients with cardiovascular disease. The ACT range for the general population is 120 to 140 seconds.
During a CPB procedure heparin will typically be administered to the patient to inhibit clot formation so as to achieve ACT values of greater than 480 seconds. Bull, B., et al., J. Thorac. Cardiovasc. Surg., 69:685-689, 1975. At the conclusion of the surgical procedure the heart lung machine is disconnected and there is no further need to inhibit blood clot formation. While the previously infused heparin would eventually leave the patient's body, one can see that it is medically imperative to restore the patient's blood clotting ability to prevent uncontrolled bleeding. The normal blood clotting state is called clinical hemostasis.
In order to achieve hemostasis, it is common to administer a substance known as protamine to the heparinzed patient. Protamines are simple strongly basic proteins of relatively low molecular weight. These proteins are water soluble, not coagulated by heat and yield only amino acids, chiefly arginine when hydrolyzed.
Protamine is a naturally occurring material and is commercially available to the medical profession as an extract from certain fish (salmon) tissue. The purity and therefore the physiological potency of commercial protamine preparations, for reasons not well understood, have been shown to vary from batch to batch. Protamine is dispensed on a weight basis. Protamine, while of different chemistry than heparin, also has the property of prolonging the blood clotting time in humans.
Heparin and protamine are reactive with each other on a stoichiometric basis. Heparin is an anionic substance and protamine is a cationic substance. When the two substances are mixed in blood (either in vivo or a test tube) they react quantitatively to form a neutral (and physiologically inactive) entity. Medical personnel therefore infuse protamine at the conclusion of, e.g., a CPB procedure, to neutralize heparin in patient's blood and restore normal, baseline blood clotting ability.
Protamine, however, as previously discussed, is itself an anticoagulant and if excess protamine is infused, hemostasis will not be achieved. Further complications can result from the fact that protamine may be toxic to some individuals--Horrow, J., Anesth. Analg., 64:348-361, 1985. Protamine is also reportedly capable of inducing an allergic response in certain patients--Sharath, M., et al., J. Thorac. Cardiovasc. Surg., 90:86-90, 1985.
Surgical teams performing thoracic surgery typically follow one of three procedures to estimate the dosage of protamine required to neutralize the heparin circulating in the patient's blood. One method is to administer protamine based on a ratio of protamine to total heparin infused during the procedure. As previously discussed, the physiological potency of heparin and of protamine vary from batch to batch and one can foresee the potential for inaccuracies.
Another method employs a procedure based on the heparin dose--response curve described by Bull, B., et al., J. Thorac. Cardiovasc Surg,, 69:685-689, 1975. At the end of cardiopulmonary bypass the concentration of heparin remaining in the patient's blood is determined by correlation of the ACT to a dose-response curve of ACT vs. heparin concentration constructed prior to bypass. Once the blood heparin concentration is known, an empirical protamine to heparin ratio is used to calculate a protamine dose which will neutralize the patient's heparin and restore clinical hemostasis.
At the present time the preferred clinical method is heparin vs. protamine titration in vitro wherein varying amounts of liquid protamine are added to heparinized blood to determine the amount of protamine required to normalize whole blood clotting times measured by the Activated Clotting Time (ACT) test. The clinical use of a protamine titration assay based on ACT technology has been limited due to the lack of a convenient and accurate protamine assay.
Irrespective of the availability of the ACT assay, it is not possible to predict how a given patient will respond to a given heparin or protamine preparation. This is primarily due to the fact that, as currently labeled, heparin or protamine preparations do not indicate the potency of the drug in a human characterized blood specimen. Currently the accepted pharmaceutical labeling of potency is the United States Pharmacopeia (USP) designation. As noted above, the USP designation of heparin potency is the anticoagulant inducing effect of heparin in a substrate of animal (sheep) citrated plasma. Conversely, protamine potency is the neutralization of heparin in this same substrate. This is referred to as the heparin neutralizing potency. There are two obvious deficiencies of this system. First the non-human substrate used does not behave the same as human material. Secondly, the substrate is citrated plasma and, for the aforementioned reasons, plasma has not proven an effective substitute for human blood as a tool to predict anticoagulant response. In sum, due to the complexity of the coagulation mechanism, the prior art methods and substances have very serious drawbacks.